Bioweapon-detecting fibrous-network products and methods for making same

ABSTRACT

Methods are disclosed for producing a bioweapon-sensitive fibrous-network product, wherein the subject products exhibit a color change in response to exposure to a biological agent (or portion thereof) as used in a biological weapon. Also disclosed are fibrous-network products that contain units of biopolymeric material that impart a color change to the products in response to exposure to a biological agent (or portion thereof) as used in a biological weapon.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional Application No.60/379,537, filed May 9, 2002.

FIELD

The present disclosure relates, inter alia, to fibrous-network productssuch as paper products, woven-fabric products, and products that arecomposites of woven and non-woven (paper) products. More specifically,the disclosure pertains to such products that exhibit a color change inresponse to contact with a biological-weapon agent (or a portion of abiological-weapon agent), and to methods for producing suchfibrous-network products.

BACKGROUND

Biological weapons (“bioweapons”) include bacteria, viruses, and toxinsthat are dispersed deliberately in an environment to cause disease ordeath to humans, animals, or plants in the environment. Examples ofbioweapons include Bacillus anthracis, which causes anthrax, Yersiniapestis, which causes plague, and Variola major, which causes smallpox.Bioweapons also include “biotoxins,” which are toxins produced bycertain biological organisms. Exemplary biotoxins are botulinum toxin,produced by the bacterium Clostridium botulinum, and ricin isolated fromcastor oil seeds. Western counter-proliferation agencies currentlyrecognize 23 types of bacteria, 43 types of viruses, and 14 types ofbiotoxins as potential bioweapons.

To an aggressor, bioweapons are appealing because: (a) they are directedindiscriminately to populations, (b) they leave infrastructure intact,(c) they usually are effective in very small amounts, (d) they can beproduced at low cost in a short period of time, and (e) their detectionis difficult. Because of these properties of bioweapons, there isgrowing concern that bioweapons will become the preferred weapon of massdestruction.

Recent activities by terrorists have targeted the U.S. Postal System byusing common envelopes as vehicles for delivering anthrax spores.Whereas, before the current outbreak of bioterrorism-related anthrax,only 18 cases of inhalational anthrax had been reported in the UnitedStates in the 20th century, ten cases of bioterrorism-relatedinhalational anthrax have now occurred. Because the U.S. Postal Servicecurrently handles an estimated 239 billion items of mail per year, therisk is high that another disease outbreak will result from envelopesdeliberately contaminated with anthrax or other bioweapon. The risk iseven larger for mail and parcel services worldwide.

The U.S. Postal Service has issued a set of guidelines for evaluatingsuspicious mail and parcels. These include use of excessive postage,lopsidedness or oddly shaped, marked with restrictive endorsements (suchas “Personal” or “Confidential”), having protruding wires, strangeodors, or stains, or denoting a city or state in the postmark that doesnot match the return address. However, current technology can respond tobioterrorism only if such a threat is suspected or known to be imminent.Presently, there is no known practical way in which to screen all mailfor bioweapons in a systematic manner.

SUMMARY

Disclosed herein are, inter alia, methods for producingbioweapon-sensing fibrous-network products that address the needssummarized above. Such a bioweapon-sensing fibrous-network productprovides, for example, an early and unambiguous alert to mail-handlingpersonnel and mail recipients that a bioweapon (or portion thereof) hasat least contacted the product, and possibly other items and equipmentin the postal media. As used herein, a “fibrous-network product” is anyproduct formed as a network of constituent fibers, which encompassesnon-woven fabrics (generally termed “paper products” herein), wovenfabrics (generally termed “woven products” herein), composites ofmultiple non-woven fabrics, composites of multiple woven fabrics, andcomposites of woven and non-woven fabrics.

In contrast to bioweapon detectors that are printed on the surface ofpaper products (e.g., as discussed in U.S. Pat. No. 6,306,598), thebioweapon-sensing fibrous-network products disclosed herein containunits of a bioweapon-sensing agent dispersed throughout at least aportion of the thickness dimension of the product. Thus, not only ismuch of the bioweapon-sensing agent shielded by fibers of the productfrom the direct physical insults inherent in, for example, mail-sortingand delivery processes, but also the agent can detect exposure to abioweapon (or portion thereof) on both the inside and outside surfacesof the fibrous-network product.

As defined herein, a “paper product” is not limited to such productsmade exclusively from cellulose fibers. “Paper product” encompasses anyof various products prepared by laying down a mat of fibers from a fluid(liquid or gas) suspension of fibers. The fibers can be cellulosic(e.g., obtained from wood pulp or other cellulosic fiber such as cottonfibers, rayon fibers, etc.), non-cellulosic (e.g., glass or polymeric),proteinaceous (e.g., silk, fur, or felt), or a mixture of one or more ofthese various types of fibers. Similarly, a woven product is not limitedto such products made from cellulose fibers. The fibers can becellulosic, as noted above, non-cellulosic, proteinaceous, or a mixtureof one or more of these various types of fibers.

To incorporate the bioweapon-sensing agent through the thicknessdimension of a paper product, units of the bioweapon-sensing agent aredispersed in a fluid suspension of the fibers used to form the paperproduct, and are formed together with the fibers in the paper product.The incorporation can extend through the length and width dimensions ofthe paper product, or be limited to certain zone(s) (e.g., stripe(s)) onthe paper product. In addition, a second fluid suspension (containingfibers and units of a second bioweapon-sensing agent) can be applied toa paper product already formed from a first fluid suspension (containingfibers, which can be the same or different from the fibers in the secondfluid suspension, and optionally units of the second or a firstbioweapon-sensing agent). In the resulting composite, eachbioweapon-sensing agent is incorporated throughout at least a portion ofthe thickness of the composite product.

For making paper products, the bioweapon-sensing agent desirably is aform that can be readily dispersed in fluid suspension with fibers usedto form the paper product. Any of various “units” of thebioweapon-sensing agent can be used that meet this criterion. Forexample, and not intending to be limiting, the units can be particles ofany of various configurations such as microspheres, membrane fragments,fibers, microtubular, or generally irregular. A particularly desirableconfiguration of the bioweapon-sensing agent is biopolymeric liposomes.Due to their spherical shape and extremely small dimensions,biopolymeric liposomes are readily dispersible in fluid suspension witha population of fibers suitable for making a paper product, and areextremely durable while retaining sensitivity to contact with one ormore specific bioweapon agents. A suspension of these liposomes iscombined with a “slurry” or other fluid suspension of fibers and mixedto form a composite mixture. The composite mixture is formed into apaper product comprising the liposomes and fibers, wherein the liposomesare intermingled with the fibers throughout the thickness dimension ofthe paper product. The biopolymeric liposomes can be immobilized insolid-support particles that are dispersible in a fluid suspension ofthe fibers and subsequently formed into a paper product. Thesolid-support particles can be, for example, sol-gel particles. In amore specific embodiment, the biopolymeric liposomes comprise an entity(“ligands”) that is sensitive to contact with one or more specific typesof bioweapon bacteria or spores. For example, the liposomes (and thusregions of the paper product containing the liposomes) exhibit a colorchange upon contact with anthrax bacteria and/or spores.

Also disclosed herein are paper products that contain biopolymericliposomes, as summarized above, intermingled with fibers throughout thethickness of at least a portion of the paper product. The paper productscan be, e.g., units of cardboard or paper. The cardboard or paper can beconfigured, for example, as a box, an envelope, a mailing tube, a label,a mailbox liner, a mail bag, or a stamp.

In another embodiment, the composite mixture of fibers andbioweapon-sensing agent are applied to an existing woven fabric andallowed to form a mat on the surface of the woven fabric or a mat thatis partially or completely intermingled with fibers of the woven fabric.

In yet another embodiment, fibrous threads are spun from a suspension offibers and bioweapon-sensing agent, yielding threads having thebioweapon-sensing agent interspersed throughout the thickness dimensionof the threads. The threads subsequently are formed into a non-wovenfabric, a woven fabric, or a composite fabric.

Although fibrous-network products as disclosed herein have especialutility for use in making mail enclosures, it will be understood thatthe fibrous-network products generally have utility in any of variousapplications in which detection of bioweapon agents is advantageous.

The foregoing and additional features and advantages of the inventionwill be more readily apparent from the following detailed description,which proceeds below.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a schematic diagram of an embodiment of a process for making apaper product in which a bioweapon-sensing agent is dispersed in thethickness dimension of the paper product.

DETAILED DESCRIPTION

I. Abbreviations

-   UV ultraviolet-   XPS x-ray photoelectron spectroscopy-   PDA diacetylene monomer-   PCA pentacosadiynoic acid monomer-   DCDA docosadynoic acid-   TRCDA tricosadiynoic acid-   SA-PDA sialic acid-derived PDA-   OTS octadecyltrichlorosilane-   VOC volatile organic chemical-   CR calorimetric response-   EDC ethylcarboiimide hydrochloride-   AFM atomic force microscope-   NHS N-hydroxysuccinimide    II. General Description of Several Embodiments

Disclosed herein are methods for producing fibrous-network products thatexhibit a color change in response to contact with one or more specifictypes of bioweapons (or portions thereof). By way of example, thesefibrous-network products are useful for alerting mail-handling personneland mail recipients after a bioweapon (or portion thereof) has at leastcontacted the product, and possibly contaminated other items in thepostal media or mail-sorting equipment. The color-changed portion of theproduct can be configured so as to provide mail-handling personnel andmail recipients with an unmistakable indication of the contamination.This, in turn, facilitates the taking of appropriate measures to verifythe contamination, contain the contamination, and/or decontaminate theaffected item and other possibly affected items and equipment asnecessary. Thus, the bioweapon-sensing fibrous-network productsdisclosed herein facilitate early and reliable detection, containment,and decontamination of bioweapons transmitted through the mail, and canprovide reassurance both to postal employees and to mail recipientswhenever contamination has not occurred.

The bioweapon-sensitive fibrous-network products disclosed herein haveseveral advantages over the bioweapon-detectors of the prior art. Inparticular, unlike bioweapon-detectors that are printed on the surfaceof paper products (e.g., as discussed in U.S. Pat. No. 6,306,598 toCharych et al.), the bioweapon-sensing fibrous-network productsdisclosed herein contain the bioweapon-sensitive agent dispersed amongconstituent fibers throughout at least a portion of the thickness of theproduct. Thus, not only is the bioweapon-sensing agent shielded byfibers from the direct physical insults inherent in the mail-sorting andmail-delivery processes, but also the sensitive agent is available fordetecting exposure to a bioweapon (or portion thereof) on both theinside and outside surfaces of the product.

In any of various particularly advantageous embodiments, thebioweapon-sensing fibrous-network product comprises biopolymericliposomes. The biopolymeric liposomes can be made from a variety ofself-assembling monomers such as diacetylene. The liposomes generallyare prepared by sonication. (New, Liposomes: A Practical Approach,Oxford University Press, Oxford, pp. 33-104 [1990]), although any ofvarious other dispersing techniques or methods can be used. In general,a solution of the self-assembling monomers is prepared. At least most ofthe solvent is removed. The residual solute is resuspended in an aqueousliquid, and the suspension is sonicated to suspend the monomers in theaqueous liquid. During this suspension, the monomers associate with oneanother to form liposomes. After liposomes having a desired median sizeand configuration are formed, sonication is halted, and the monomers areallowed to polymerize to form the biopolymeric liposomes. Biopolymericliposomes and their production are discussed in greater detail insubsections III and IV, below.

The self-assembling monomers of the biopolymeric liposomes arechemically linked to other molecules (e.g., ligands), that are specificin their ability to interact with one or more types of bioweapon agentcontacting the liposomes. Hence, contact of a ligand with a bioweaponagent (or portion thereof) causes the biopolymeric liposome to exhibit acolor change. By way of example, a ligand can be used that is sensitiveto contact with anthrax organisms or spores, wherein biopolymericliposomes comprising the ligands exhibit a color change in response tosuch contact. Further with respect to the example, the“anthrax-sensitive” ligand can be a substrate for a laccase, which is amulti-copper-containing enzyme that catalyzes oxidative conversion of avariety of substrates, including phenols, polyphenols, and aromaticamines. Specific substrates include vanillic acid, syringic acid, and2-2′-azino-bis(3-ethyl-benzthioazoline-6-sulfonic acid). Incorporatingone or more laccase substrates into ligands attached to biopolymericliposomes yields liposomes that are sensitive to anthrax exposure. Inother embodiments, the ligand is an antibody specific for a bioweaponagent or molecule or portion of a bioweapon agent or molecule.Bioweapon-detecting ligands and their attachment to the self-assemblingmonomers are discussed in greater detail in subsection VI, below.

As an alternative to biopolymeric liposomes, the biopolymeric materialcan have any of various different forms, wherein particles ofbiopolymeric material generally are termed “units” of biopolymericmaterial. The units can be configured as sheets, tubules, or other“regular” shape, or can have any of various “irregular” configurations.For incorporation with fibers from which a fibrous-network product is tobe made, the units of biopolymeric material desirably are fragmented orotherwise comminuted to an appropriate mean particulate size. Particularadvantages of forming the units of biopolymeric material as liposomesare the very high dimensional uniformity with which liposomes can beformed, and the high strength and robustness of the very small sphericalconfiguration of liposomes.

The biopolymeric materials may further comprise one or more “dopants,”which are used to alter and/or to optimize desired properties of thebiopolymeric materials. Such properties can include, but are not limitedto, colorimetric response, color, sensitivity, durability, amenabilityto immobilization, temperature sensitivity, and pH sensitivity of thebiopolymeric materials. For example, adding sialic acid-deriveddiacetylene monomers to liposomes comprising ganglioside and PDA causesthe liposomes to exhibit increased colorimetric sensitivity to exposureto low levels of particular bioweapons. Dopants, and their inclusion inbiopolymeric liposomes, are discussed at greater length in subsection V,below.

Units of the biopolymeric material can be immobilized on or in any of avariety of solid supports, including, but not limited to, polystyrene,polyethylene, Teflon®, silica gel beads, hydrophobized silica, mica,filter paper (e.g., Nylon®, cellulose, and nitrocellulose), glass beadsand slides, gold, and various separation media such as silica gel,Sephadex®, and other chromatographic media. Immobilization of thebiopolymeric materials can improve, for example, their stability,shelf-life, calorimetric response, color, or other desired properties.In some embodiments, the biopolymeric materials are immobilized in asol-gel. Immobilization of the biopolymeric liposomes is discussed ingreater detail in subsection VII, below.

Manufacture of a bioweapon-sensing fibrous-network product isexemplified by manufacture of a bioweapon-sensing paper product, inwhich a suspension of units of biopolymeric material is added to a fluidsuspension of fibers and mixed to form a composite mixture. Thecomposite mixture is formed into a paper product comprising the units ofbiopolymeric material and the fibers, wherein the units of biopolymericmaterial are intermingled with the fibers throughout the thicknessdimension of the paper product. In some embodiments, the units ofbiopolymeric material are intermingled with the fibers throughout alength or a width dimension of the paper product, thereby forming abioweapon-sensitive stripe or stripes in the paper product. In otherembodiments, the units of biopolymeric material are intermingled withthe fibers throughout the length and width dimensions of the paperproduct. The paper product can be, e.g., individual or bound sheets ofpaper, cardboard or paper configured as a box, an envelope, a tube(e.g., mailing tube), a label, a mailbox liner, a mail bag, or a stamp.Formation of a bioweapon-sensitive paper product is discussed in greaterdetail below, in subsection VIII.

Whenever the bioweapon-sensitive fibrous-network product contacts abioweapon agent (or portion thereof), for example during mail-sorting ormail-delivery, the product exhibits a color change in response to thecontact. For example, in some embodiments, the color change is from blueto red. In some embodiments, the entire fibrous-network product changescolor. In other embodiments, the color change is localized to aparticular region of the product, for example, a stripe on an envelope.

The bioweapon-sensitive fibrous-network product can be configured sothat the color change is visible to product-handling personnel and/orrecipients. Thus, for example, a postal employee or mail recipient canbe assured that, for example, if an envelope does not exhibit a changedcolor, then the envelope has not contacted a detectable dose of thebioweapon to which the envelope is sensitive.

If a bioweapon-sensitive fibrous-network product has contacted adetectable dose of the corresponding bioweapon agent, and the productreveals a characteristic color change, then product-handling personnelor recipients can initiate appropriate steps to verify thecontamination, contain the contamination, and/or decontaminate theproduct and the surrounding environment as necessary. Detection of abioweapon with the bioweapon-sensitive fibrous-network productsdisclosed herein is discussed at greater length in subsections IX and X,below.

III. Biopolymeric Materials

Any of various monomers are suitable for forming the biopolymericmaterial. The monomers can be self-assembling or may require catalysisfor assembly. Such monomers include, but are not limited to, acetylenes,carbohydrates, diacetylenes (e.g., 5,7-docosadiynoic acid,5,7-pentacosadiynoic acid, and 10,12-pentacosadiynoic acid), alkenes,thiophenes, polythiophenes, imides, acrylamides, methacrylates,vinylether, malic anhydride, urethanes, allylamines, siloxanes,polysilanes, anilines, pyrroles, polyacetylenes,poly-(para-phylenevinylene), poly-(para-phylene), and vinylpyridinium.The monomers as incorporated into lipids can be homopolymers or mixedpolymers. Furthermore, the monomers can have any of various head groupsincluding, but not limited to, carboxyl groups, hydroxyl groups, primaryamines, amino acid derivatives, and hydrophobic groups. The monomers canbe chemically linked to any of various other molecules (e.g.,bioweapon-specific ligands).

In some embodiments, lipid monomers comprising diacetylene are used asself-assembling monomers for forming the biopolymeric material. Avariety of diacetylene-containing lipid monomers can be used including,but not limited to, 5,7-docosadiynoic acid (5,7-DCDA),5,7-pentacosadiynoic acid (5,7-PCA), and 10,12-pentacosadiynoic acid(10,12-PCA).

In other embodiments, the biopolymeric material comprisesglycopolythiophenes or other polythiophenes that exhibit a color change(e.g., a red-shift) upon binding with one or more specific bioweaponagents. The polythiophenes can be used as such or can include any ofvarious specific receptors, such as polypeptides, lipids, orcarbohydrates. An example of such modified polythiophenes iscarbohydrate-modified polythiophene (glycopolythiophenes). Theglycopolythiophenes, for example, can be further modified for enhancedspecificity to a specific bioweapon agent. Modification can includeattachment of other carbohydrate lipids, dopants, protein ligands,antibody ligands, and hexokinase ligands.

In order to increase or decrease the sensitivity of the biopolymericmaterial, the position of a chromophore on the polymer backbone of thebiopolymeric material can be varied. For example, bioweapon-sensitivityof a biopolymeric liposome may be increased by moving diacetylene groupsof the constituent diacetylene monomers closer to the interfacial regionof the liposome. In addition, shorter or longer chain lengths of PDA canaffect the sensitivity of the biopolymeric material for detection of aparticular bioweapon.

The length of a carbon chain positioning a head group a specificdistance from the polymer backbone of the biopolymeric material isdependent on the position of the polymerizable group in the monomermolecules from which the biopolymeric material is formed. In the case ofbiopolymeric liposomes formed from diacetylene monomers, for example, adiacetylene group positioned from between the 18-20 carbon positions tothe 3-5 carbon positions in the monomers, from between the 10-12 carbonpositions to the 4-6 carbon positions, or about the 5-7 carbon positionscan enhance sensitivity. The production protocol for the monomersdetermines the final position of the diacetylene group in the monomersused for making the biopolymeric material.

The total carbon-chain length in the monomer also influences the levelof sensitivity of biopolymeric material made from the monomer, althoughgenerally to a lesser extent than the position of the polymerizablegroup in the carbon-chain of the monomer. Specifically, a shorter chainlength typically confers greater bioweapon-sensitivity. Usefulcarbon-chain lengths range from between C₁₂ to C₂₅, although both longerand shorter chain lengths can be used. In some embodiments, the monomercarbon-chain length is C₂₀ to C₂₃.

IV. Production of Biopolymeric Liposomes as a RepresentativeBiopolymeric Material

As noted above, the biopolymeric material used in the present disclosuredesirably comprises biopolymeric liposomes. These liposomes desirablyare prepared using sonication. (New, Liposomes: A Practical Approach,Oxford University Press, Oxford, pp. 33-104 [1990]), although any othermethod usable for generating liposomes alternatively can be employed. Asolution of self-assembling monomers, either alone or in associationwith a desired ligand, is prepared. At least most of the solvent isremoved, and the solute is resuspended in an aqueous liquid. Thesuspension is sonicated to form the liposomes. The self-assemblingmonomers in the liposomes are allowed to polymerize.

Liposomes have been described in numerous articles and have beenreviewed in texts such as New (supra), and Rosoff, Vesicles, MarcelDekker, New York, [1996]). As noted above, liposomes typically are morestable and robust than films or other structures made of the samebiopolymeric material.

In general, liposomes are prepared by dispersing amphiphilic moleculesin an aqueous medium. In an aqueous suspension, the constituentamphiphilic molecules of liposomes typically remain in a liquid phase.In an aqueous suspension liposomes can have any of various shapes suchas spheres, ellipsoids, squares, rectangles, and tubules (but usuallyare spherical). In most of these configurations the surface of aliposome is in contact with the aqueous carrier liquid only. Ifliposomes are “dried” (i.e., if sufficient carrier liquid is removed),they may lose their shape and no longer exist in a liposomal state(i.e., the constituent molecules disassociate and thus no longer areconfigured as “liposomes”).

Hence, the molecules making up the liposomes desirably are polymerizedwith each other in each liposome, which prevents dissociation andprovides substantial liposome stability under various environmentalconditions. Polymerization desirably is performed by exposure toultraviolet (UV) light, which is convenient. Alternatively,polymerization can be performed by application of another suitablesource of bonding energy, such as a catalyst or temperature change.

In a biopolymeric liposome, the polymer backbone of the biopolymer willabsorb different energies of light, and hence will exhibit a differentcolor, depending upon the degree of polymerization of the biopolymer,the presence of other molecules in the liposomes, and the accessibilityof solvent molecules to the biopolymers in the liposome. Suchdifferential absorption of light provides the liposomes with a desiredcolor. The degree of polymerization can be manipulated to provide theliposomes selectively with one or more desired properties. For example,cooling a suspension of liposomes before initiating polymerization cansubstantially reduce polymerization (cooling to room temperature beforeinitiating polymerization can inhibit UV-initiated polymerizationcompletely). On the other hand, polymerization can be initiated readilyin liposomes pre-cooled to 4° C., wherein a subsequent increase intemperature to room temperature has no adverse affect on polymerization.In any event, polymerization usually is evidenced by appearance of, or achange in color of the liposome suspension, e.g., appearance ofabsorption of light having a wavelength of approximately 630 nm.

As discussed below, binding of a bioweapon molecule or structuresituated proximally to the biopolymeric liposome also is detectablethrough an observable color change of the biopolymeric liposomes (andhence of the fibrous-network product with which the liposomes areincorporated).

V. Dopants

The biopolymeric materials disclosed herein may comprise one or moredopants. Dopants serve to alter and optimize certain properties of thebiopolymeric materials. Such properties include, but are not limited to,colorimetric response, color, sensitivity, durability, robustness,amenability to immobilization, temperature sensitivity, and pHsensitivity. For example, the addition of sialic acid-deriveddiacetylene monomers to liposomes comprising ganglioside and PDA confersto the liposomes a dramatic increase in colorimetric sensitivity of theliposomes with respect to the detection, of low levels of certainbioweapons. This improvement in colorimetric response using dopant isextremely beneficial whenever a non-doped biopolymeric material producesa colorimetric response that is too weak to be of practical use.

As noted above, dopants can change the color of the biopolymericmaterial before and/or after contact with a bioweapon agent to which thebiopolymeric material is sensitive. For example, a color change ofliposomes is from blue to red (before and after contact, respectively).Other color changes achievable by doping are blue to orange, purple tored, purple to orange, green to red, and green to orange. For example,glutamine-substituted PDA produces very dark blue (i.e., almost black)biopolymeric material. Green biopolymeric material can be produced byapplication of cycles of annealing (i.e., heating to approximately 80°C.) and cooling (i.e., to room temperature) prior to polymerization. Theadvantage of altering the color profile (before and after contact) isthat fibrous-network products can be made that start at a desired colorbefore contact and exhibit a specific color in response to contact witha specific bioweapon. Multiple dopants can be combined in a singlebiopolymeric material preparation.

The most appropriate concentration of dopant incorporated with thebiopolymeric material depends upon the particular properties desired inthe fibrous-network product. For instance, certain applications mayrequire compromised sensitivity in favor of long shelf-life ordurability under rigorous field conditions. The acceptable concentrationrange of dopant is from a concentration having no affect to aconcentration that would excessively disrupt the stability of thebiopolymeric structures.

By way of example, molar percentages of dopant normally range from 0.01%to 75%. However, certain embodiments may require a percentage of dopantgreater than 75% or lower than 0.01%. In some embodiments, a desirablerange for dopant is 2%-10%, more desirably about 5%.

In selecting appropriate incorporation methods for the dopant, there areseveral competing considerations. For example, when producing liposomesby conventional sonication, proper incorporation of large and/or complexdopants typically requires relatively slow and gentle sonication, whichmay be difficult to accommodate in an industrial process. Also,sonication is most effective whenever only a relatively lowconcentration of dopant is to be incorporated. Point-probe sonicationallows the incorporation of much higher concentrations of dopant over ashorter period of time (typically minutes). However, point-probesonication typically is limited to incorporation of small tointermediate-sized dopant molecules. The temperature at which dopantincorporation is performed is selected based on the particular liposomeparameters desired. A practitioner will be able to select the foregoingand other parameters such as pH, choice of diluents, and other factorsbased on the particular system and desired characteristics of thebiopolymeric material.

VI. Ligands

The biopolymeric materials disclosed herein desirably further compriseone or more types of ligands that act as recognition sites in thebiopolymeric materials for specific bioweapon agents. Upon theinteraction of a bioweapon molecule or other unit thereof with a ligandor ligands on a molecule of biopolymeric material, a disruption of thepolymer backbone of the biopolymeric material occurs, resulting in adetectable chromatic transition (color change) of the biopolymericmaterial.

A ligand can be linked to a self-assembling monomer or to a biopolymericmaterial formed from such monomers, by: (1) a linker attached to theself-assembling monomer, (2) a direct linkage to the monomer withoutusing a linker, (3) being incorporated into the biopolymeric materialprior to or during polymerization of the monomers, or (4) being attachedto the biopolymeric material following polymerization (e.g., by linkingligands to constituents of the biopolymeric material that contain headgroups that can be bonded directly to the ligands or via other means).

The ligand can be any of a wide variety of materials. The main criterionis that the ligand have an affinity at least for the bioweapon ofchoice. Appropriate ligands include, but are not limited to, peptides,carbohydrates, antigens, molecular-recognition complexes, antibodies, orany of various organic molecules that bind to receptors. Thebiopolymeric material can be formed from combinations of ligand-linkedand unlinked monomers, thereby optimizing a desired colorimetricresponse (e.g., 5% ligand-linked dicosadynoic acid [DCDA] and 95% DCDAhaving no linked ligands). Additionally or alternatively, multipleligands can be incorporated into the biopolymeric materials. As is clearfrom the broad range of ligands that can be used, an extremely diversegroup of bioweapons can be detected.

By way of example, the selected ligand is specific for any of variouspathogenic organisms and biotoxins, including, but not limited to,anthrax, cryptococcosis, Escherichia coli, Haemophilus influenzae,brucellosis (undulant fever), coccidioidomycosis (San Joaquin Valley ordesert fever), psittacosis (parrot fever), Yersinia pestis (the BlackDeath of the 14th Century), tularemia (rabbit fever), malaria, cholera,typhoid, bubonic plague, cobra venom, shellfish toxin, botulinum toxin,saxitoxin, ricin, smallpox, Shigella flexneri, S. dysenteriae (Shigellabacillus), Salmonella, Staphylococcus enterotoxin B, hemorrhagic fever,Venezuelan equine encephalitis, Histoplasma capsulatum, pneumonicplague, Rocky Mountain spotted fever, dengue fever, Rift Valley fever,diphtheria, melioidosis, glanders, tuberculosis, infectious hepatitis,encephalitides, blastomycosis, nocardiosis, yellow fever, typhus,tricothecene mycotoxin, aflatoxin, Q fever, and severe acute respiratorysyndrome (SARS).

From the present disclosure, one skilled in the art will be able toselect and incorporate an appropriate ligand(s) with biopolymericmaterials as disclosed herein. For example, methods for adding any of adiverse range of substituent groups (e.g., carbohydrates, proteins,nucleic acids, and other chemical groups) to lipid molecules are wellknown in the art. By way of example, the carboxylic acid group on theterminus of a lipid molecule can be easily modified to form an ester, aphosphate ester, an amino group, an ammonium ion, a hydrazine, apolyethylene oxide, an amide, or any of various other compounds. Any ofthese chemical groups can serve as a site for linking a carbohydrate, aprotein, or other chemical group. E.g., a carboxylic acid group on alipid molecule can be directly linked to a protein by first convertingthe carboxylic acid group to the corresponding activated ester, followedby reaction of the activated ester with an amine group on a proteinmolecule to form an amide linkage of the protein to the lipid.

Thus, any of various protein molecules can be attached to biopolymericliposomes, such as on the surface of the liposomes, thereby providingthe liposomes with “protein” ligands. Protein ligands include, but arenot limited to, peptides, lipoproteins, glycoproteins, enzymes,receptors, channels, and antibodies. Whenever a molecule or other unitof a corresponding bioweapon binds to the ligand, the polymer backboneof the respective biopolymeric material is subjected to a disruptionsufficient to cause the biopolymeric material to exhibit a color change.

For example, Bacillus anthracis organisms and spores have laccaseactivity. The presence of these organisms and spores can be detected byreaction of laccase with a laccase substrate. Laccases aremulti-copper-containing enzymes that catalyze oxidation of varioussubstrates, including phenols, poly-phenols, and aromatic amines.Specific laccase substrates include vanillic acid, syringic acid, and2-2′-azino-bis(3-ethyl-benzthioazoline-6-sulfonic acid). Introducing oneor more of these laccase substrates as ligands into the biopolymericmaterials of the present disclosure confers a sensitivity of thebiopolymeric materials to contact with anthrax organisms and spores. Oneof skill in the art will be able to identify other ligands that areuseful in detecting other bioweapons, such as antibodies or enzymesubstrates specific for the Variola major virus that causes smallpox.

VII. Immobilization of Biopolymeric Materials

Units of biopolymeric material can be immobilized on any of variety ofsolid supports, including, but not limited to, polystyrene,polyethylene, Teflon®, silica gel, hydrophobized silica, mica,filter-paper medium (e.g., Nylon®, cellulose, and nitrocellulose), glass(e.g., glass beads), gold particles, and various separation media suchas Sephadex™ or other chromatographic or electrophoretic media.Immobilization of the biopolymeric material in this manner can improvethe stability, shelf-life, calorimetric response, color, and/or otherdesired properties of the fibrous-network product comprising thebiopolymeric material.

In some embodiments, the units of biopolymeric material are immobilizedin a sol-gel. For example, biopolymeric liposomes are robust underaqueous conditions and ambient temperatures, but can degrade whenexposed to an organic solvent and/or high temperature. Incorporating thebiopolymeric liposomes into a sol-gel provides a simple method ofimmobilizing the liposomes without causing any significant structuremodification of the liposomes, thereby creating even more robuststructures that can be easily incorporated into a fibrous-networkproduct and/or formed into any desired size or shape.

An exemplary sol-gel can be prepared by sonicatingtetramethylorthosilicate, water, and hydrochloric acid until asingle-phase solution is obtained. Any of various other metal oxides canbe used as an alternative to tetramethylorthosilicate, so long as theselected oxide facilitates the subsequent entrapment of the units ofbiopolymeric material in the sol-gel and forms sol-gel particles thatare sufficiently transparent to allow a color change of entrappedbiopolymeric material to be visible or detectable. Such metal oxidesinclude, but are not limited to, silicates, titanates, aluminates,ormosils, and others.

Following sonication, the solution is suitably buffered, and asuspension of units of biopolymeric material, produced as describedabove, is added to and mixed with the buffered sol-gel suspension. Thesuspension is poured into a desired molding structure and allowed tocongeal. Many types of molding structures can be used to produce gels ofany desired size and shape, including, but not limited to, beads, rods,and fibers. Gelation can take place under ambient conditions, or at anyother suitable temperature that facilitates the production of thedesired sol-gel.

The gelling conditions of the sol-gel preparation can be optimized byvarying the gelling temperature, the gel material, and/or the dryingconditions to generate a sol-gel having a desired pore size. Control ofpore size also can be achieved by suitably varying the degree ofcross-linking of the gel. Pore sizes can range from nanometers tohundreds of nanometers or greater. Some gels allow size-selectivescreening of undesired material while maintaining bioweapon access tothe ligand.

After forming the sol-gel, the gel mass is fragmented or comminuted toform particles or fibers suitable for incorporation with other fibersinto a fibrous-network product. In an example, DCDA biopolymericliposomes are incorporated into sol-gel particles, following the sol-gelprocedure as described above. Gelation occurs within a few minutes,yielding a sol-gel exhibiting a violet color.

VIII. Incorporation of Units of the Biopolymeric Material into aFibrous-Network Product

Incorporation of units of biopolymeric material (which may or may not beimmobilized into a solid support) into a fibrous-network product isexemplified by forming a paper product comprising the biopolymericmaterial. Formation of the paper product is advantageously performedusing a paper-making machine. Alternatively, the paper product can bemade by any of various processes by which a fibrous mat is laid downfrom a fluid suspension (gas or liquid suspension) of fibers and otherparticles to be incorporated with the fibers into the paper product.

A representative paper-making process is depicted in FIG. 1, in whichmany of the components will be readily understood by persons of ordinaryskill in the relevant art, and hence are not described in detail below.The process begins with a pulper 1. A furnish 2 (fibers to be used tomake the paper product) is placed in the pulper 1 along with a suitablecarrier liquid 3 (typically water). The carrier liquid 3 can passthrough a UV-irradiator 4 to eliminate or reduce bacterial contaminationof the carrier liquid before the carrier liquid enters the pulper 1. Thecarrier liquid 3 desirably has a pH in the range of pH 4-8 as measuredby a pH monitor 5. The proper pH can be achieved as a result of thenatural pH of the carrier liquid or by appropriate buffering. Thesuspension of fibers (“slurry”) proceeds from the pulper 1 through aslurry tank 6 and machine chest 7. Meanwhile, a suspension of units ofthe biopolymeric material (e.g., a suspension of biopolymeric liposomes)is stored in a biopolymer-unit tank 13. The slurry dispensed from themachine chest 7 enters the pre mix tank 8 in which the slurry is mixeduniformly with the suspension of units of the biopolymeric material fromthe biopolymer-unit tank 13 to form a composite mixture. To ensure thatthe contents of the pre-mix tank 8 are uniformly mixed for use, thepre-mix tank 8 is provided with a nitrogen-sparger 9, an agitator 10, arecirculation loop with inline mixer 11, and an ultrasonic transducer12. When the composite mixture has a desired concentration of fibers andbiopolymeric material, and a desired viscosity, the composite mixturepasses from the recirculation loop 11 through a discharge valve 15 to amix tank 16. The temperature of the mix-tank 16 is controlled by atemperature controller 17 to minimize temperature fluctuations of thecomposite mixture. From the mix tank 16, the composite mixture isdischarged through a screen 18 to a head box 19. From the head box 19,the composite mixture is deposited onto a paper former 20. The paperformer 20 is essentially an endlessly moving screen that forms acontinuous mat of the fibers and units of biopolymeric material retainedon the screen. The carrier liquid passes through the screen, whereinsuch passage typically is facilitated by application of a vacuum 21. Themat desirably is routed through a dryer 22 comprising multiple rollers23 in a temperature-controlled housing. As the mat passes through thedryer 22, HEPA-filtered air 24 is directed at the mat to remove residualcarrier liquid and thus form the paper product. Desirably, to protectthe biopolymeric materials in the paper product, the drying temperatureis regulated to less than about 55° C. For example, the dryingtemperature can be room temperature. To facilitate drying, the dryer 20can include one or more vacuum-drying pads over which the paper productpasses during passage through the dryer. If necessary, the paper productis subjected to ionizers 25 during passage through the dryer to avoidstatic buildup on the paper product. Following drying, the paper productcan be rolled using a winder 26. The recovery system 27 collects and/orrecycles from the paper former 20 and/or the screen 18.

The finished paper product produced by the apparatus of FIG. 1 comprisesunits of biopolymeric material (e.g., biopolymeric liposomes)intermingled with fibers throughout a thickness dimension of the paperproduct. In some embodiments, the finished paper product comprises thebiopolymeric material intermingled with fibers throughout a length orwidth dimension of the paper product, resulting in a stripe or stripesof bioweapon-sensitive biopolymeric material in the paper product. Inother embodiments, the finished paper product comprises units ofbiopolymeric material intermingled with fibers throughout the length andwidth dimensions of the paper product, resulting in a uniformdistribution of the bioweapon-sensitive biopolymeric material throughoutall dimensions of the paper product. In yet other embodiments, the paperproduct can include any of various other dyes, pigments, and/oradditives that can be added to a paper product to achieve desiredphysical characteristics of the paper product. The paper product canhave a thickness and/or weight in the realm of “paper” or a greaterthickness and/or weight, such as card stock, cardboard, or the like.

Formation of a fibrous-network product comprising units of thebiopolymeric material also is exemplified by forming a woven fabric ofthreads or yarns made from the fibers. For example, units ofbiopolymeric material are added to a suspension of suitable fibers fromwhich threads or yarns are spun. As the fibers are spun from thesuspension, they incorporate the biopolymeric material through thethickness dimension of the threads or yarns. The threads or yarns arethen woven into a fabric containing the units of biopolymeric material.Alternatively, fibrous threads or yarns can be dipped into or sprayedwith a suspension of biopolymeric material, and the resulting threads oryarns woven into a fabric. Note that both warp and woof of such fabricneed not be of the same threads. Indeed, one thread (e.g., woof) cancontain the bioweapon-sensing agent while the other (e.g., warp) doesnot. Further alternatively, to a woven fabric (which may or may notcontain a bioweapon-sensing agent) can be applied a non-woven mat offibers that contain a bioweapon-sensing agent (or alternatively, asecond bioweapon-sensing agent that is different from a first such agentin the woven fabric). Further alternatively, a fabric is formed byfelting fibers to which the bioweapon-sensing agent has been added. Inall these various embodiments, units of biopolymeric material containingone or more bioweapon-sensing agents are intermingled with fibers of theresulting fibrous-network products through at least a portion of thethickness dimension of the product. A finished fabric created byspinning, weaving, or felting the fibers and particles described hereincomprise units of biopolymeric material intermingled with fibers.

In some embodiments, the finished fabric comprises the biopolymericmaterial intermingled with fibers throughout a length or width dimensionof the fabric, resulting in a stripe or stripes of bioweapon-sensitivebiopolymeric material in the fabric. In other embodiments, the finishedfabric comprises units of biopolymeric material intermingled with fibersthroughout the length and width dimensions of the fabric, resulting in auniform distribution of the bioweapon-sensitive biopolymeric materialthroughout all dimensions of the fabric. In yet other embodiments, thefabric can include any of various other dyes, pigments, and/or additivesthat can be added to a fabric to achieve a desired physicalcharacteristic.

After forming the fibrous-network product, it can be subjected toconventional downstream processing to form it into any of variousconfigurations for use, such as office-paper sheets, greeting-cardblanks, boxes, envelopes, mailing tubes, labels, mailbox liners, mailbags, or stamps, for example. Woven fabrics and composites comprisingthe biopolymeric materials described herein are particularly useful forcreating durable bioterrorism-detecting fibrous-network products. Forexample, such a product may be used to form a mailbox liner or mail bag.

IX. Detection of a Bioweapon Agent with Bioweapon-SensitiveFibrous-Network Products

A fibrous-network product as described above exhibits a color changewhen brought into contact with a specific bioweapon(s) to which theconstituent biopolymeric material is sensitive. Such contact causes acorresponding disruption of the polymer backbone of the biopolymericmaterial. The resulting color change can be detected using any ofvarious methods. For example, a color change is observable simply byvisual inspection of the product. Fibrous-network products amenable tovisual inspection can be used easily by an untrained person.Alternatively or in addition to visual detectability, a color change inthe product can be detected using a colorimetric scanner. Thecolorimetric scanner can be used for directly detecting a color changeor for detecting a specific fluorescence wavelength produced by theproduct when irradiated with suitable UV light. For example, a barcodereader incorporated into mail-sorting machinery can be configured todetect fluorescence in paper products exposed to a specific bioweapon.

In certain embodiments, a fibrous-network product exposed to a specificbioweapon exhibits a color change from blue (non-exposed) to red(exposed). Other embodiments exhibit other color(s) depending uponvarious factors discussed above such as type and amount of dopants. Thefibrous-network product can be configured to exhibit the color changelocally (e.g., in a stripe) or overall. For example, the paper stockused to form envelopes can be provided with a sensitive stripe or thelike that is present in each and every envelope. Contact with a specificbioweapon causes a respective color change in the stripe rather than theentire envelope.

Under most conditions of use, the color change exhibited by thefibrous-network product upon exposure to a respective bioweapondesirably is visible to the user. If the product is used formail-related purposes, the bioweapon-exposed product would bediscernible by mail-handling personnel and/or mail recipients anddistinguishable from non-contaminated items. Hence, a postal employee ormail recipient encountering an item of mail not exhibiting a colorchange is assured that, for example, the item has not been exposed to adetectable dose of anthrax organisms or spores. On the other hand, if acolor change has occurred in the mail item, then the person noticing thecolor change can take prompt action to limit and remove thecontamination, such as inspecting other items of mail and limitingcontact with suspect mail-sorting and mail-delivery equipment asrequired.

X. Other Uses

Even though the ligands incorporated into biopolymeric materials used infibrous-network products as discussed herein desirably are selectedbased on their respective sensitivity to one or more specific bioweaponagents, it is comprehended that similar ligand-attachment technology canbe used to form fibrous-network products that are “sensitive” to contactwith any of various other analytes of interest.

The following examples are provided so as to provide furtherdescription, and are not intended to be limiting in any way.

EXAMPLES Example 1 Preparation of Biopolymeric Material

Self-assembling monomers to be incorporated into biopolymeric liposomesare dissolved in respective solvents (e.g., chloroform for diacetylenesand methanol for gangliosides such as G_(M1) used in this example). Anyof various other solvents can be used, including, but not limited to,benzene, hexane, and ethylacetate. The solutions are prepared and storedin opaque containers to prevent excessive contact with light. Thesolutions are combined to yield a lipid mixture of 5% by mole of G_(M1)and 95% by mole of diacetylenes (total lipid content of approximately 2μmol). After preparing the combined solution, the solvents areevaporated (e.g., by rotary evaporation or using a stream of nitrogengas).

To the dried solute is added sufficient de-ionized water to produce a1-15 mM suspension of lipid. The suspension is sonicated to form thelipids into liposomes. The duration of sonication depends upon the typeand power output of the sonicator employed (e.g., 15-60 minutes using aFisher probe sonicator model 300 operated at 50% output, fitted with amicrotip). Unless sonication provides sufficient intrinsic heating, thesuspension is heated during sonication to heat the suspension to atemperature above the phase-transition temperature of the lipids in thesuspension (typically 30-90° C.). The resulting liposome suspension isfiltered through a microporous filter and either cooled to 4° C. forstorage or immediately subjected to polymerization conditions.Desirably, prior to polymerization, oxygen is removed from thesuspension by nitrogen sparging, and nitrogen sparging is continuedduring polymerization.

Polymerization is performed by exposure to ultraviolet (UV) light (e.g.,254 nm) at a sufficient dose (J/cm²) of light to initiate and propagatethe polymerization. Polymerization is continued in this manner for atime sufficient to achieve the desired properties (e.g., color,polymerization degree) of the liposomes.

Example 2 Incorporation, Optimization, and Properties of Dopants

Each bioweapon-sensitive fibrous-network product made as disclosedherein desirably is made with respective concentrations of monomer,dopant, and ligand sufficient to achieve the desired performance. Therespective concentrations can encompass wide ranges (e.g., 5-15%ligands, 0-95% PDA, and 0-95% dopant). The percent of each componentdepends upon the physical characteristics of the fibrous-network productas well as the desired stability and sensitivity of the product to oneor more specific bioweapons. Also, as noted above, more than one dopantcan be used.

I. Incorporation of Dopant into Biopolymeric Material

A diacetylene dopant (including amino acid substituent groups) anddiacetylene monomers are dissolved in chloroform and transferred to areaction container. The organic solvent is evaporated using N₂ gas, andwater is added to bring the lipid concentration to approximately 1 mM.The resulting mixture is sonicated to form liposomes. Sonication isperformed at approximately 80° C. to facilitate formation of theliposomes and continued for a time sufficient to clarify the mixture.The resulting liposome suspension is immediately filtered (while hot)through a microporous filter to remove particulate impurities. Thefiltered suspension either can be stored at 4° C. until use or(desirably) used immediately for polymerization.

Polymerization is performed using UV light as noted above, yieldingliposomes or other units of biopolymeric material exhibiting a deep-bluecolor.

II. Properties of Derivatized Diacetylene Dopants

If the amino acid substituent groups in the diacetylene dopant moleculesare hydrophobic, then the dopant can be used for reducing the solubilityof the biopolymeric material in aqueous media and for reducing thestability of the liposomes. These derivatized PDAs can be useful forfine-tuning the stability and sensitivity of the paper productsincorporating the biopolymeric material (stability and sensitivity aredirectly related to each other). If the units of biopolymeric materialcontain several types of dopants (e.g., PDAs including hydrophobic aminoacid substituent groups and PDAs including hydrophilic amino acidsubstituent groups), then the stability of the biopolymeric material canbe tailored for specific environmental conditions. For example, certainconditions may require high stability with a tolerable reduction insensitivity. Under most conditions a balance between sensitivity andstability is desired.

Molecules of diacetylene monomers can be linked to acidic and/or basichead groups to increase the solubility of the PDA in a particularliquid(s). Ordinarily, PDA is not water-soluble; a relativelyhydrophobic organic solvent is necessary for dissolving PDA.Unfortunately, many hydrophobic organic solvents destroy biologicalmolecules. By linking an acidic or basic head group to a PDA molecule,the solubility of the PDA in a relatively hydrophilic solvent(s) isgreatly enhanced. Also, in many instances, biopolymeric materialsincluding these modified PDAs produce brighter colors and hence are moresensitive than liposomes lacking them.

In another example, amine-coupling histidine to PDA creates a dopantthat, when incorporated into biopolymeric material, causes the materialnot only to exhibit a ready color change when exposed to a specificbioweapon but also to be regeneratable. I.e., ordinarily polymerizedPDAs exhibit color changes upon exposure, but cannot be reused. Thenear-neutral pK_(a) of the head group provided by the linked histidineallows reuse.

In yet another example, amine-coupling fluorescent head groups to PDAs,and incorporating the PDAs in biopolymeric material yields biopolymericmaterial that exhibits fluorescent properties. Whenever units of suchmaterial, incorporated into a fibrous-network product, are brought intocontact with a specific bioweapon, the units of biopolymeric material(and thus the fibrous-network product) exhibit a change in fluorescence.This type of product is readily detectable by automated equipment and issuitable for certain covert operations involving mail surveillance, forexample.

Example 3 Attachment of Ligands

Ligands desirably are covalently linked (i.e., linked by covalent bonds)to the head groups of self-assembling monomers used for forming thebiopolymeric material. Ligands also can be covalently linked to othermaterials including polymerized materials. For example, proteins andantibodies can be linked with one or more amine and/or thiol linkages tosurfaces of liposomes. Ligands also can be non-covalently incorporatedinto the biopolymeric material (e.g., ganglioside molecules incorporatedinto liposome membranes).

The synthesis technology for making self-assembling monomers thatinclude any of various head-group functionalities is known in the art.Exemplary head-group functionalities include, but are not limited to,esters, ethers, amino groups, amides, thiols, or combinations thereof.Alternately, ligands can be incorporated by surfactants intoself-assembling monomers without covalent linkage (e.g., incorporationof gangliosides and lipoproteins into liposome-membrane regions havingmultiple hydrophobic moieties).

Specific applications are described below to illustrate the broad rangeof ligands that can be associated with the biopolymeric material. Theseexamples are intended to be exemplary only, and not limiting in any way.

I. Sialic Acid

In this example, sialic acid is attached as a ligand to diacetylenemonomers. Any of several synthesis methods can be used to form thelinkage, especially synthesis methods generally useful for attachingcarbohydrates to the biopolymeric materials. In a first example, 2.7mmol PDA in chloroform is reacted with 3 mmol N-hydroxysuccinimide and 3mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).The reaction is allowed to proceed with stirring for 2 hours followed byevaporation of the chloroform. The residue is extracted with diethylether and water. The organic layer is dried with magnesium sulfate(MgSO₄) and filtered. Residual diethyl ether is evaporated, yieldingN-succinimidyl-PDA (NHS-PDA). To a solution of NHS-PDA in chloroform isadded ethanolamine (3 mmol), followed by triethylamine (2.5 mmol). Themixture is stirred for two hours at room temperature. The chloroform isevaporated, and the residue is purified by chromatography in silica gel(2:1 EtOAc:hexane, R_(f)=0.15), yielding purifiedN-(2-hydroxyethyl)-PDA.

The purified N-(2-hydroxyethyl)-PDA is dissolved in chloroform (1.3mmol), and tetraethylene glycol diamine (6.60 mmol) in chloroform isadded dropwise, with stirring, over a period of 30 minutes. The solutionis stirred for an additional 30 minutes, and the solvent is evaporated.The residue is dissolved in EtOAc and extracted twice with water. Theorganic layer is dried with MgSO₄, and the chloroform removed byevaporation. The resulting extract is purified by chromatography insilica gel (20:1 CHCl₃:MeOH, R_(f)=0.20), yielding purified N-(11-amino-3,6,9-trioxyundecanyl)-PDA.

Approximately equal volumes of acetic anhydride and a cooled solution ofethyl-5-N-acetyl-2,6-anhydro-3,5-dideoxy-2-C-(2-propenyl)-D-erythro-L-manno-nonoate(1.30 mmol) in 1.7 mL of pyridine are mixed together under nitrogen,with stirring. The reaction is allowed to warm to room temperature,followed by removal of the pyridine by application of reduced pressureat ambient temperature. The resulting viscous oil is solidified byrepeated evaporation from toluene and flash chromatographed over silica,with ethylacetate as an eluent, to yield purifiedethyl-5-N-acetyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-C-(2-propenyl)-D-erythro-L-manno-nonoate.

A solution ofethyl-5-N-acetyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-C-(2-propenyl)-D-erythro-L-manno-nonoate(0.72 mmol in acetone) is cooled to −78° C. in the absence of moisture.Ozone is aspirated into the solution until a persistent blue color isobtained. Excess ozone is purged using O₂ gas, and the solution iswarmed to 5° C. Excess Jones' reagent is added to obtain a persistentrust-orange color, and the solution is further warmed to roomtemperature. Ethanol is added slowly to consume excess O₂ and ozone. Theresulting green precipitate is filtered and washed with acetone. Thefiltrate is evaporated in vacuo and dissolved in ethyl acetate. Thesolution is extracted with saturated aqueous NaHCO₃, and the aqueousphase is acidified with concentrated HCl and extracted with methylenechloride. The methylene chloride extract(s) are dried, filtered, andevaporated in vacuo to yieldethyl-5-N-acetyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-C-(aceticacid)-D-erythro-L-manno-nonate.

Ethyl-5-N-acetyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-C-(aceticacid)-D-erythro-L-manno-nonate (0.35 mmol) is added, under nitrogen, toa 5° C. solution of NHS (0.50 mmol) and EDC (0.50 mmol) in chloroform.The mixture is warmed to room temperature with stirring, diluted withchloroform, and washed with 1 N HCl (aq.), with saturated (aq.) sodiumbicarbonate, and with saturated (aq.) sodium chloride. The organic layeris dried over MgSO₄, filtered, and evaporated to yieldethyl-5-N-acetyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-C-(N-succinimidylacetate)-D-erythro-L-manno-nonoate.

Ethyl-5-N-acetyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-C-(N-succinimidylacetate)-D-erythro-L-manno-nonoate(0.22 mmol) and N-(11-amino-3,6,9-trioxyundecanyl)-PDA (0.24 mmol) aredissolved in chloroform. The mixture is sealed and stirred for a timesufficient for reaction to progress to completion. The solution isdiluted with chloroform and washed with sodium-chloride-saturated 1N HCl(aq.), with saturated (aq.) sodium bicarbonate, and with saturated (aq.)sodium chloride. The organic layer is dried over MgSO₄ and filtered.Residual solvent is evaporated, and the resulting semi-solid is flashchromatographed over silica (20:1 CHCl₃:MeOH) to yield purifiedethyl-5-N-acetyl-4,5,8,9-tetra-O-acetyl-3,5-dideoxy-2-C-[(N-11′-(PDA)-3′,6′,9′-trioxyundecanyl)acedamido]-D-erythro-L-manno-nonoate.

PDA with sialic acid substituent groups is formed by dissolvingethyl-5-N-acetyl-4,5,8,9-tetra-O-acetyl-3,5-dideoxy-2-C-[(N-11′-(PDA)-3′,6′,9′-trioxyundecanyl)acedamido]-D-erythro-L-manno-nonoate (0.19 mmol) in a solution of 8:1(v/v) water:methanol containing sodium hydroxide sufficient to renderthe solution basic. The solution is stirred, followed by addition ofion-exchange resin (Biorad AG 50W-X4H⁺ form) is added until the solutionis acidic. The solution is filtered and the filtrate evaporated invacuo, producing PDA derivatized with sialic acid.

II. Carbohydrates

Carbohydrates (which can include sialic acid) can be modified by athree-step procedure to produce N-allyl glycosides. The N-allylglycosides can be linked to other molecules (e.g., PDA) using knownchemical-synthesis methods. Thus, a broad range of carbohydrates can beincorporated into biopolymeric material (thereby allowing detection of abroad range of bioweapon-related analytes).

The carbohydrates can be obtained from oligosaccharides, in whichinstance the oligosaccharides are dissolved in neat allyl amine (watermay be added if necessary and does not adversely affect the yield)producing a 0.5-0.1 M solution of the oligosaccharides that, in theallyl amine, react to form amino glycosides. Upon completion of thereaction, the solvent is removed by evaporation and the remaining solidsare washed several times with toluene and evaporated to dryness. Thewashed solid is chilled, and a solution of 60% pyridine, 40% aceticanhydride is added to yield a solution containing five hundredmole-percent excess of acetic anhydride. The reaction is protected frommoisture, stirred, and allowed to warm to room temperature. The solventsare removed by evaporation, and the residue is dissolved in toluene anddried by evaporation several times. The residue is purified by flashchromatography to yield respective peracetylated NAc-allyl-glycosideforms of the free sugars.

The peracetylated NAc-allyl glycosides are dissolved in anhydrousmethanol to yield a 0.1-0.01 M solution. A small amount of 1 N NaOMe inMeOH is added and allowed to react. Sufficient Dowex® 50 resin (H⁺ form)is added to neutralize the base, and the solution is filtered andevaporated to dryness (purification by recrystallization can beconducted if desired). The products are the N-allyl glycosylamide formof the carbohydrates.

The reaction scheme described in this example can be used to produce therespective N-allyl glycosylamide forms of a variety of carbohydrates,including, but not limited to, glucose, NAc-glucosamine, fucose,lactose, tri-NAc-chitotriose, sulfo Lewis analog, and sialyl Lewisanalog. Skilled artisans will appreciate the general applicability ofthis method to the attachment of a broad range of carbohydrates todiacetylene lipids.

III. Ganglioside G_(M1)

Ganglioside G_(M1) is exemplary of incorporation of a ligand withself-assembling monomers without covalent attachment of the ligands tothe monomers. Ganglioside G_(M1) is introduced by combining a solutionof methanol-dissolved ganglioside G_(M1) with chloroform-dissolved PDA.The subsequently dried solution includes a ganglioside that contains ahydrophobic region facilitating its incorporation into self-assemblingsurfactant structures. Thus, when the dried solution is resuspended inwater, structures are formed that contain a mixture of ganglioside andPDA. Liposomes and other structures are produced from the resuspendedmixture as described in Example 1. Although the ganglioside does notcontain a polymerizable group, the ganglioside becomes embedded in thepolymerized matrix created by the cross-linking of the diacetylenes.Similar methods can be used for the incorporation of other ligandscontaining hydrophobic regions (e.g., transmembrane proteins andlipoproteins) into the biopolymeric material.

IV. Proteins

NHS-PDA produced as described above, thiol-linked PDA, and othercompounds provide functional groups useful for attaching proteins andantibodies. The NHS or thiol-linked monomers are incorporated into theliposomes and polymerized, thereby providing NHS or thiol functionalgroups that provide surface-reaction sites on the liposomes useful forcovalently linking proteins and antibodies to the liposomes using knownchemical-synthesis reactions. In another embodiment, a hydrazidefunctional group can be attached to PDA, allowing subsequent linkage ofthe PDA to aldehydes and ketone groups of proteins and antibodies. Thus,a broad array of proteins and antibodies can be attached to thebiopolymeric material. Specific examples are provided below. Theseexamples are not intended to be limiting.

A. Hexokinase

NHS-PDA lipid is synthesized as described above. In brief,10,12-pentacosadiynoic acid is dissolved in CHCl₃, to whichN-hydroxysuccinimide (NHS) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added.After stirring at room temperature, CHCl₃ is removed by evaporation. Theresidue is extracted with EtOAC and water. The organic layer is driedwith MgSO₄ and filtered, followed by solvent removal. The solids aredissolved in and recrystallized from CHCl₃.

Yeast hexokinase suspension is spun in a microcentrifuge to removesaturated ammonium sulfate. The protein is resolubilized in 0.1 Mphosphate buffer (pH 8.0) to approximately 1 mg/mL, and dialyzed againstthe same buffer. PDA-containing liposomes are incubated in thehexokinase solution at 4° C. Prolonged incubation results in decreasedcolor intensity. The liposomes are rinsed with water and immersed into0.1 M ethanolamine to terminate the linking reaction. The liposomes arerinsed again with water and dried. Polymerization is conducted byirradiation with UV light.

B. Antibodies

Diacetylene is filtered to remove insoluble impurities (e.g.,polymerized forms) and converted chemically to NHS-PDA as describedabove. Appropriate amounts of NHS-PDA and other forms of PDA derivatives(e.g., dopants or ligands) are mixed together at the desired molarratio. The suspension is dried, and water is added to bring the totalconcentration of lipid to approximately 1 mM. The solution is sonicateduntil the liquid is clarified and filtered hot through a microporousfilter. The filtrate can be stored at 4° C. until time of use.

Prior to cross-linking, 0.1 M phosphate buffer (pH 8.5) is added to theliposome suspension. A solution of antibody molecules in a similarbuffer is then added and allowed to react at 4° C. Excess antibody isremoved by either centrifugation or dialysis. If centrifugation is used,the pellet is re-sonicated gently under ice-bath-temperature conditions.After linking of the antibody molecules to the sonicated material,polymerization is conducted as described for liposomes in Example 1.

Antibodies also can be attached to biopolymeric material by hydrazides.In some embodiments, this manner of antibody attachment may be preferredto NHS-coupling because NHS may react at the Fab′ region of theantibody, blocking the ability of the antibody to bind to analytes. Thehydrazide method results in attachment of the Fc region of theantibodies to the biopolymeric material, leaving the analyte-bindingregion of the antibody available for binding to analytes. In thehydrazide method, however, hydrazide-PDA-lipids are produced, along withunpolymerized material.

A stock antibody solution is washed, using Centricon® 50 filters, byadding an equal volume of 123 mM sodium citrate (pH 5.5). The solutionis centrifuged, and filtration-washing is repeated. A volume of theantibodies in citrate buffer are oxidized by incubating with sodiumperiodate at room temperature. The reaction is quenched by addition ofN-acetylmethionine. Next, a mixture of liposomes, citrate buffer, water,and oxidized antibodies is incubated at room temperature to link theantibodies to the liposomes. Unlinked antibodies are removed from theliposomes by filtration-washing with Tris buffer (pH 9.0) andcentrifugation. After washing the sample is diluted as required withTris buffer to make a 0.2 mM (or less) liposome solution.

Any of various other techniques can be used for linking or otherwiseassociating various molecules to the biopolymeric material.Self-assembling monomers associated with ligands can have modified chainlengths or may consist of double or multiple chains. These variouscombinations of ligands and monomers provide a broad array ofbiopolymeric materials appropriate for the interaction with a broadrange of analytes, with the desired colorimetric response, selectivity,and sensitivity.

Example 4 Sol-Gel Immobilization of Biopolymeric Material

In this example a silica sol is prepared by sonicatingtetramethylorthosilicate (TMOS), water, and 0.04 N aqueous hydrochloricacid under ice-bath-temperature conditions until the solution is onephase. This solution is termed a “sol.” Chilled MOPS buffer solution(50% v/v) is added to the acidic sol under ice-bath-temperatureconditions to retard gelation.

Various materials are appropriate for producing silica sols, including,but not limited to, any tetraalkoxysilane or organically modified silane(e.g., ormosil). Additionally, tetraethylorthosilicate (TEOS),methyltriethoxysilane (MeTEOS), aryl silsesquioxanes, and other metaloxides are useful for producing silica sols.

For producing the liposomes, a polymerized liposome solution (producedas described in Example 1) is added to the buffered sol. The mixture canbe poured into molds, formed into films, or otherwise allowed to gel atroom temperature under sealed conditions. Gelation occurs within a fewminutes, resulting in transparent, monolithic units of gel. If the gelcontains p-PDA liposomes, then coloration is evident.

Example 6 Colorimetric Analysis

Colorimetric changes of the biopolymeric materials made as describedabove can be detected by simple visual observation, including by anuntrained observer such as an at-home user. Other techniques useful fordetecting a color change of the fibrous-network product are set forthbelow.

I. Visible Absorption Spectroscopy

In some embodiments, it may be preferred to obtain quantitative datapertaining to color changes of the fibrous-network product, or to recordsubtle changes or faint responses of the product that are undetectableby the human eye.

One manner of performing detections at increased accuracy is byspectroscopy. For example, spectroscopy can be performed of absorptionof visible light by the product. For PDA-containing liposomes, acolorimetric response (CR) can be quantified by measuring a percentchange in absorption at 626 nm (imparting blue color to the material)relative to an absorption maximum.

The visible-light absorption B⁰ of a biopolymeric material that has notyet been exposed to an analyte can be expressed as:B ₀ =I ₆₂₆/(I ₅₃₆ +I ₆₂₆)wherein the various “I” variables denote absorption intensity at therespective subscripted wavelengths. The absorption B_(a) of thebiopolymeric material after exposure to the analyte is expressed as:B _(a) =I ₆₂₆/(I ₅₃₆ +I ₆₂₆)The calorimetric response (CR) of a liposome solution is defined as thepercentage change in the absorption spectrum (B) upon exposure toanalyte:CR=[(B ₀ −B _(a))/B ₀](100%)

Terms

To facilitate an understanding of the present disclosure, a number ofterms and phrases are defined below:

“Amino acid” refers to an organic compound having both an amino group(NH₃) and a carboxylic acid group (COOH) appropriately ionized dependingupon the pH of the environment of the amino acid. Amino acids are theessential components of protein molecules.

“Amphiphilic” refers to a molecule having a polar, water-soluble groupattached to a non-polar, water-insoluble hydrocarbon chain.

“Analyte” refers to any material that is to be analyzed. Analytesinclude, but are not limited to, ions, molecules, antigens, bacteria,compounds, viruses, cells, antibodies, and cell parts.

“Antibody” refers to a glycoprotein evoked in an animal by exposure to acomplementary immunogen (antigen). An antibody demonstrates specificbinding to the respective immunogen, or, more specifically, to one ormore epitopes contained in the immunogen.

“Antigen” refers to any molecule, molecular group, or structure that isrecognized by at least one antibody. An antigen must contain at leastone “epitope” (i.e., the specific biochemical unit capable of being“recognized” by the antibody in a manner resulting in binding of theantibody to the unit).

“Aqueous” refers to a liquid solution or mixture containing water as asolvent or carrier, respectively.

“Bacteria” and “bacterium” refer to any of various prokaryoticorganisms, including organisms within various phyla in the KingdomProcaryotae. The terms encompass all microorganisms commonly regarded asbacteria, including Mycoplasma, Chlamydia, Actinomyces, Streptomyces,and Rickettsia. The terms also include cocci, bacilli, spirochetes,spheroplasts, protoplasts, etc.

“Biological weapon” or “bioweapon” refers to any of various bacteria,viruses, and toxins that is or can be dispersed deliberately to causedisease or death to humans, animals, or plants, or other biologicalorganisms. Examples of biological weapons include Bacillus anthracisthat causes anthrax, Yersinia pestis that causes plague, and Variolamajor that causes smallpox. Biological weapons also include biotoxins,which any of various poisons produced by certain biological organisms,such as botulinum toxin, produced by the bacterium Clostridiumbotulinum, and ricin, isolated from castor oil seeds.

“Biopolymeric material” refers to a material composed of one or moretypes of polymerized biological molecules (e.g., lipids, proteins,carbohydrates, and combinations thereof). Biopolymeric materialsinclude, but are not limited to, films, vesicles, liposomes,multilayers, aggregates, membranes, and solvated polymers (e.g.,polythiophene aggregates such as rods and coils in solvent).Biopolymeric materials can also comprise unpolymerized molecules.

“Calorimetric” refers to the measurement of heat.

“Carbohydrate” refers to a class of molecules including, but not limitedto, sugars, starches, cellulose, chitin, glycogen, and similarstructures. Carbohydrates can exist as components of glycolipids andglycoproteins.

“Channel” refers to an integral membrane protein that provides for theregulated transport of a specific ion, or ions, across a membrane.

“Chromatic transition” refers to a change of a molecule or material thatresults in an alteration of visible-light absorption by the molecule ormaterial. Chromatic transition can refer to a change in light absorptionof a sample, whereby a detectable color change of the sample isassociated with the transition. Detection of a chromatic transition canbe accomplished through various means including, but not limited to,visual observation and spectrophotometry.

“Chromophoric” refers to the property of a chemical group that permitsselective light absorption and results in a characteristic coloration.

“Composite product” refers generally to a class of fibrous-networkproducts that include or are made from multiple non-woven fabrics,multiple woven fabrics, or woven and non-woven fabrics integratedtogether.

“Covalent bond” refers to the linkage of two atoms by the sharing of oneor more pairs of electrons by the atoms.

“Derivatize” refers to producing or obtaining a chemical from anothersubstance by chemical reaction.

“Diacetylene monomer” refers to a hydrocarbon molecule containing twoalkyne linkages (i.e., carbon/carbon triple bonds).

“Direct calorimetric detection” refers to a detection of a color changewithout the aid of an intervening processing step (such as conversion ofa color change into an electronic signal that is processed by aninterpreting device). This term encompasses visual observation (e.g.,observation performed directly with the human eye) as well as detectionby simple spectrophotometry.

“Dopant” refers to a molecule that, when added to a biopolymericmaterial, changes a property of the material. Candidate propertiesinclude, but are not limited to, calorimetric response, color,sensitivity, durability, amenability to immobilization, temperaturesensitivity, and pH sensitivity of the material. Dopant materialsinclude, but are not limited to, lipids, cholesterols, steroids,ergosterols, polyethylene glycols, proteins, peptides, or any othermolecule (e.g., surfactants, polysorbate, octoxynol, sodium dodecylsulfate, zwitterionic detergents, decylglucoside, deoxycholate,diacetylene derivatives, phosphatidylserine, phosphatidylinositol,phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol,phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide,cerebroside, lysophosphatidylcholine, D-erythroshingosine,sphingomyelin, dodecyl phosphocholine, N-biotinylphosphatidylethanolamine, polyethylene glycol, peptide detergents,3-[-(cholamidopropyl)dimethylammonio]-1-propanesulfonate, and othersynthetic or natural components of cell membranes) that can beassociated with a membrane (e.g., liposomes).

“Encapsulate” refers to encompassing, encasing, or otherwise associatingtwo or more materials with each other in a manner such that anencapsulated material is immobilized within or onto a respectiveencapsulating material.

“Enzyme” refers to a biological molecule or molecular aggregate thatcatalyzes a chemical or biological reaction. Enzymes are typicallyproteins, but “enzyme” can also comprise any of various short peptides,RNAs, ribozymes, antibodies, and other molecules.

“Furnish process” refers generally to a paper-making process.

“Glycolipid” refers to a lipid containing a carbohydrate group.

“Glycoprotein” refers to a protein containing a carbohydrate group.

“Head group” refers to a molecular groups situated at an end of amolecule (e.g., a carboxylic acid group at the end of a fatty acid).

“Homopolymer” refers to a polymer formed by the bonding together ofmultiple units of a single type of molecular species (i.e., a single“monomer”).

“Hydroxyl group” refers to the univalent radical or group, OH, which isa characteristic component of bases, certain acids, phenols, alcohols,carboxylic and sulfonic acids, and amphoteric compounds.

“Immunogen” refers to any molecule, compound, or structure that inducesthe production of antibodies by an antibody-producing organism. Bydefinition, an immunogen must contain at least one epitope.

“Interfacial region” refers to the region between two plane surfaces orfaces, particularly two membrane faces.

“Ligand” refers to any ion, molecule, molecular group, or othersubstance that binds to another molecule or molecular structure, usuallyin a specific manner. Examples of ligands include, but are not limitedto, ions, charged molecules, peptides, carbohydrates, nucleic acids(e.g., DNA and RNA), antibodies, or any of various molecules that bindto receptors.

“Lipid” refers to any of various compounds that are soluble in ahydrophobic organic solvent. Lipids include, but are not limited to,fats, waxes, steroids, sterols, glycolipids, glycosphingolipids(including gangliosides), phospholipids, terpenes, fat-soluble vitamins,prostaglandins, carotenes, and chlorophylls.

“Lipoprotein” refers to any of various conjugated proteins in which atleast one of the components is a lipid.

“Liposome” refers to a spherical lipid complex that can be induced tosegregate out of aqueous media.

“Mixed polymer” refers to a material comprised of two or more types ofpolymers.

“Molecular-recognition complex” refers to any molecule, molecular group,or molecular complex that is capable of recognizing (i.e., specificallyinteracting with) a particular molecule. For example, the ligand-bindingsite of a receptor is a molecular-recognition complex.

“Paper product” refers generally to a class of fibrous-network productsthat are non-woven, typically made from cellulosic and/or non-cellulosicfibers laid down from a fluid suspension of the fibers. The fibers canbe obtained from pulp containing wood fibers, cotton fibers, grassfibers, and/or other natural fibers. Alternatively or in addition, thefibers can be non-cellulose polymeric, mineral, glass, or other fibers.Paper products generally are processed into leaves, sheets, rolls, orother units by deposit of the fibers from a fluid suspension of thefibers with binders and other additives as required. Paper products caninclude, but are not limited to, paper leaves (sheets), cards,envelopes, boxes, tubes, labels, mailbox liners, mail bags, and stamps.

“Paper pulp” refers to a suspension of cellulose pulp used for making apaper product.

“Peptide” refers to two or more amino acids covalently joined by peptidebonds.

“Polymer” refers to any of numerous natural and synthetic compoundscomposed of repeated linked units, each a relatively light and simplemolecule.

“Polymerizable group” refers to a chemical group that is capable ofundergoing polymerization.

“Polymerization” refers to the uniting of two or more monomers to form apolymer.

“Postal media” refers to any of various media used in the delivery ofmail and parcels. Postal media can include, but are not limited to,paper, cardboard, envelopes, boxes, mailing tubes, labels, mailboxliners, mail bags, and stamps.

“Primary amine” refers to derivatives of ammonia in which one hydrogenhas been replaced by a hydrocarbon unit. Some common primary amines areaniline, methylamine, and 1-propylamine.

“Protein” refers broadly to any of various molecules or molecularassemblies containing two or more amino acids linked together by apeptide bond. Proteins include, but are not limited to, peptides,enzymes, antibodies, receptors, lipoproteins, and glycoproteins.

“Pulper” refers to a step in a paper-manufacturing process in whichpaper pulp is suspended in water to form a slurry.

“Receptor” refers to a grouping on a macromolecule or a cell which cancombine selectively with other complementary molecules or cells (e.g.,enzyme receptors or cell surface receptors for hormones or growthfactors).

“Room temperature” refers to a temperature approximately between 20 and25 degrees centigrade.

“Self-assembling monomer” refers to molecules that spontaneouslyassociate to form molecular assemblies, such as surfactant moleculesthat associate to form surfactant molecular assemblies. Self-assemblingmonomers include single molecules (e.g., molecules of a single type oflipid) and small molecular assemblies (e.g., molecules of polymerizedlipids), that can be further aggregated (e.g., assembled andpolymerized) into larger molecular assemblies.

“Slurry” refers to a suspension of any of various particulatesubstances, such as cement, plaster of Paris, clay, or cellulose fibers,in a carrier liquid.

“Sol-gel” refers to a colloidal suspension of silica particles that isgelled to form a solid. The resulting gel is porous, and can havebiological or other material entrapped within the porous structures.

“Sol-gel material” refers to any material prepared by a sol-gel process,including the sol-gel itself and any material entrapped within thesol-gel.

“Sol-gel process” refers to any method that results in the production ofa sol-gel. The materials used to produce the sol-gel can include, butare not limited to, aluminates, aluminosilicates, titanates, ormosils(organically modified silanes), metal oxide glasses, and other metaloxides.

“Solid support” refers to a solid object or surface upon which a sampleis layered, attached or. Solid supports include, but are not limited to,glass, metals, gels, and filter paper, among others.

“Substrate,” in one sense, refers to a material or substance on which anenzyme or other reaction means acts. In another sense, it refers to asurface on which a sample grows or is attached.

“Ultraviolet irradiation” (UV radiation) refers to exposure to radiationwith wavelengths less than that of visible light (i.e., less thanapproximately 360 nM) but greater than that of X-rays (i.e., greaterthan approximately 0.11 nM). Ultraviolet radiation possesses greaterenergy than visible light and is therefore, more effective at inducingphotochemical reactions.

“Woven product” refers generally to a class of fibrous-network productsthat are made by weaving (e.g., by weaving warp and woof or by knitting)threads or yarns to form a fabric. The threads or yarns are formed of orcomprise any of the various types of fibers listed in the definition of“paper product,” above.

Whereas the invention has been described above in connection withmultiple representative embodiments, it will be understood that theinvention is not limited to those embodiments. On the contrary, theinvention is intended to encompass all modifications, alternatives, andequivalents as may be included within the spirit and scope of theinvention, as defined by the appended claims.

1-34. (canceled)
 35. The product of claim 69, wherein the network is awoven network.
 36. The product of claim 69, wherein the network is anon-woven network.
 37. The product of claim 69, wherein the network is acomposite network.
 38. The product of claim 69, wherein the color changepertains to a change in color of light in the visible spectrum.
 39. Theproduct of claim 69, wherein the color change pertains to a change incolor of fluorescence.
 40. The product of claim 69, wherein the colorchange pertains to a change in color of non-visible light.
 41. Theproduct of claim 69, wherein the units of biopolymeric material arebiopolymeric liposomes.
 42. The product of claim 69, wherein: the unitsof biopolymeric material comprise multiple types of ligands that arespecific and thus sensitive to contact with different respective typesof biological weapons; and contact with each type of biological weaponcauses a respective color change of the units of biopolymeric material.43. The product of claim 69, wherein the units of biopolymeric materialare sensitive to contact with anthrax organisms.
 44. The product ofclaim 69, wherein the units of biopolymeric material comprise a ligandsensitive to contact with a laccase.
 45. The product of claim 44,wherein the ligand comprises laccase substrates.
 46. The product ofclaim 44, wherein the units of biopolymeric material comprise ligandsthat are antibodies to at least one type of biological weapon.
 47. Theproduct of claim 69, having a configuration selected from the groupconsisting of sheets, boxes, cards, envelopes, tubes, labels, mailboxliners, mail bags, and stamps.
 48. (canceled)
 49. The product of claim74, wherein the particles of solid-support medium comprise sol-gelparticles.
 50. The product of claim 49, wherein the sol-gel particleshave an average diameter of from about one micrometer to about onemillimeter. 51-68. (canceled)
 69. A biological-weapon-sensing fibrousproduct, comprising: a network of fibers, the network having a thicknessdimension, a width dimension, and a length dimension; and units ofbiopolymeric material distributed among the fibers of the network so asto be distributed substantially similarly through the thicknessdimension of the network, wherein the units of biopolymeric materialcomprise ligands that are sensitive to contact with a biological weaponsuch that the units exhibit a color change upon contacting thebiological weapon.
 70. The product of claim 69, wherein the units ofbiopolymeric material are distributed substantially uniformly throughthe thickness dimension of the network.
 71. The product of claim 36,wherein the non-woven network is a paper.
 72. The product of claim 71,wherein the paper comprises cellulosic fibers.
 73. The product of claim72, wherein the paper comprises a mixture of cellulosic andnon-cellulosic fibers.
 74. The product of claim 69, wherein the units ofbiopolymeric material are embedded in particles of a solid-supportmedium that are distributed substantially similarly through thethickness dimension of the network.
 75. The product of claim 69, whereinthe units of biopolymeric material are distributed among the fibers ofthe network throughout the length and width dimensions of the network soas to form at least one bioweapon-sensitive zone in the fibrous product.76. The product of claim 75, wherein the units of biopolymeric materialare distributed among the fibers of the network throughout the lengthand width dimensions of the network so as to form at least onebioweapon-sensitive stripe in the fibrous product.
 77. The product ofclaim 69, wherein the units of biopolymeric material are distributedamong the fibers of the network throughout the length and widthdimensions that are coextensive with length and width dimensions of thefibrous product.
 78. The product of claim 69, configured as a non-wovenfibrous product comprising the network of fibers in which the units ofbiopolymeric material are distributed.
 79. The product of claim 69,configured as a woven fibrous product comprising the network of fibersin which the units of biopolymeric material are distributed.
 80. Theproduct of claim 69, configured as a composite of at least one fabric.81. The product of claim 80, wherein the fabric is woven or non-woven.82. The product of claim 69, further comprising a fabric to which thefibrous product is attached.
 83. The product of claim 82, wherein thefabric is a woven fabric.
 84. The product of claim 82, wherein thefabric is a non-woven fabric.
 85. The product of claim 82, wherein thefabric contains units of biopolymeric material that are sensitive tocontact with a biological weapon.
 86. The product of claim 69, whereinthe units of biopolymeric material comprise units of a first type ofbiopolymeric material that is sensitive to contact with a first type ofbiological weapon that causes a respective color change of the units ofthe first type, and units of a second type of biopolymeric material thatis sensitive to contact with a second type of biological weapon thatcauses a respective color change of the units of the second type. 87.The product of claim 69, wherein: the fibrous product has a firstsurface and an opposing second surface; and the fibrous product isconfigured to reveal detection of the biological weapon on both thefirst and second surfaces.
 88. The product of claim 69, wherein units ofthe biopolymeric material are embedded in fibers that are distributedsubstantially similarly through the thickness dimension of the network.